Our project aims to boost bacterial cellulose (BC) production in Komagataeibacter xylinus and utilize it as a water-retaining substrate to enhance seedling survival rates in desert environments, ameliorating the issue of desertification. We successfully knocked out the gdh gene, which encodes glucose dehydrogenase responsible for converting glucose into gluconate and overexpressed the pgi gene from E. coli. The genetically modified Komagataeibacter xylinus exhibited growth characteristics similar to the wild type, reaching a plateau on day 4, while BC production increased by 227%, reaching 4.91 g/L. After further optimizing the culture conditions, BC production increased to approximately 5.34 g/L. Testing demonstrated that BC slows soil moisture loss, improves seedling drought tolerance, and enhances survival.

Gene editing chassis selection: Komagataeibacter xylinus
Our goal is to efficiently synthesize bacterial cellulose (BC) using synthetic biology and selecting an optimal production strain is a fundamental step. BC is produced by several bacteria, including species from the genera Acetobacter, Komagataeibacter, Agrobacterium, Bacillus, Clostridium botulinum, Lactobacillus, and Bacillus glucoses [1]. Among these, Komagataeibacter xylinus is recognized for its superior cellulose-producing capacity, being one of the most efficient known strains [2]. It can rapidly synthesize cellulose through well-established metabolic pathways, achieving significantly higher yields than other species. Furthermore, Komagataeibacter xylinus secretes large quantities of high-purity cellulose extracellularly, eliminating the need for complex purification processes [3].

In addition, the genome of Komagataeibacter xylinus has been extensively studied, making it highly amenable to genetic modifications. Through metabolic engineering, it is possible to further enhance cellulose yield and optimize its properties, which enhances the strain's potential for industrial applications [3].

Knock-out gdh and Overexpression pgi from E. coli
The biosynthesis of bacterial cellulose (BC) involves three major processes: the uptake and activation of glucose to produce glucose-6-phosphate, the formation of UDP-glucose, and the polymerization of cellulose chains under the action of the cellulose synthase complex, involving several enzymatic reactions [3, 4]. Since we cannot manipulate all the related genes in this project, we consulted Professor Xu from Zhejiang University, an expert in genetic engineering. He suggested focusing on increasing the availability of glucose-6-phosphate, a key precursor in BC biosynthesis, to achieve our goal.

In a study by Jang et al. in 2019, random sampling and correlation analysis predicted that reactions involving glucose-6-phosphate isomerase (PGI) were more positively correlated with cellulose nanofiber (CNF) production rates. Their experiments confirmed that the pgi gene from E. coli significantly promoted BC production in the strain they used [4]. PGI catalyzes the conversion of fructose 6-phosphate to glucose 6-phosphate, enhancing the metabolic flow for BC production.

Most cellulose acetate-producing strains can extracellularly convert glucose into gluconic and ketogluconic acid, with membrane-bound glucose dehydrogenase (GDH) being the enzyme responsible for this conversion [5]. The production of gluconic acid reduces the available glucose in the medium, negatively affecting cellulose production. Comparative studies have identified isolates with lower GDH activity that are more efficient at producing cellulose from glucose [6].

To increase BC production, we propose knocking out the gdh gene and overexpressing a heterologous pgi gene, leading to the accumulation of glucose-6-phosphate, ultimately boosting BC production.

Plasmid selection and design
Genetic manipulation of Komagataeibacter xylinus has been challenging, with high difficulty in achieving stable transformations [2]. Due to the inherent instability of plasmids, they can be lost during bacterial propagation in the absence of selective pressure, leading to the loss of target gene expression. Therefore, while plasmids serve as ideal short-term vectors, they are not suitable for long-term solutions. To ensure more efficient and stable genetic modifications, we plan to integrate the genes directly into the chromosome of K. xylinus via homologous recombination [7]. This approach involves introducing homology arms of approximately 700 bp both upstream and downstream of the target gene, allowing for effective recombination. However, this also results in a larger insert fragment. Hence, we have selected the small-sized, high-copy number plasmid pGEM-T as a vector.

The kana gene will be inserted at the gdh locus in K. xylinus to achieve gene knockout. Additionally, to facilitate screening for positive transformants, the kana gene will be expressed under the control of the tac promoter and rrnB-T terminator.

For the overexpression of the pgi gene from E. coli, the sacB locus, which encodes levansucrase, was selected as a homologous recombination site because knocking out this gene has no metabolic effects under glucose conditions [4]. First, the kana expression cassette will replace the sacB gene locus as a selectable marker. Then, pgi will replace the kana gene, resulting in the loss of kana resistance, confirming successful gene insertion.

Cycle 1 - Build: Vector construction
To facilitate the selection of resistance genes on the genome, we first constructed an expression cassette containing the Kanamycin resistance gene (kana) flanked by homology arms, each 700-800 bp in length, on either side of the sacB gene. The upstream and downstream homology arms of the sacB gene were amplified by PCR from K. xylinus genomic DNA. The upstream flanking region was amplified using the primers sacB-5-F and sacB-5-R, while the downstream flanking region was amplified using the primers sacB-3-F and sacB-3-R. The Kanamycin resistance gene, used for antibiotic selection, was also amplified by PCR from the pET28a plasmid with the primers Km-F and Km-R. For expression, the tac promoter and rrnB-T terminator fragments were amplified from the expression vector pKK223-3 using the primers Ptac-F, Ptac-R, rrnB-T-F, and rrnB-T-R (figure 3A, 3B).

These five fragments (i.e., the upstream and downstream regions of the sacB gene, tac, rrnB, and kana) were then ligated into the pGEM-T (Easy) vector between the SalI and SacI restriction enzyme sites using T4 ligase, resulting in the pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3 plasmid. The colonies resulting were confirmed by PCR and DNA sequencing of the amplified DNA fragment (figure 3C, 3D).

Next, we amplified the pgi gene, which is 1650 bp in size, from the E. coli genome using the primers Gibson-pgi-F and Gibson-pgi-R. Simultaneously, a linearized vector fragment, excluding the kana gene, was amplified from the pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3 vector (Figure 4A). The pgi gene and the vector fragment were ligated using the Gibson assembly (ClonExpress II One Step Cloning Kit, Vazyme, China), resulting in the pGEM-sacB-5-Ptac-pgi-rrnB-T-sacB-3 plasmid. The resulting colonies were screened for ampicillin resistance and confirmed by PCR (Figure 4B).

Gene Knockout of gdh
To construct a gdh gene knock-out strain, we constructed an expression cassette containing the kana gene flanked by homology arms, each about 700 bp in length, on either side of the gdh gene. The upstream and downstream homology arms of the gdh gene were amplified by PCR from K. xylinus genomic DNA. The upstream flanking region was amplified using the primers gdh-gibson-gdh5-F and gdh-gibson-gdh5-R, while the downstream flanking region was amplified using the primers gdh-gibson-gdh3-F and gdh-gibson-gdh3-R. For expression, the fragment containing tac promoter, kana, and rrnB-T terminator were amplified from the expression vector pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3 using the primers gdh-gibson-pkr-F and gdh-gibson-pkr-R. The plasmid backbone was amplified from the same vector using the primer gdh-gibson-vector-F and gdh-gibson-vector-R (Figure 5A).

These four fragments were ligated using the Gibson assembly (ClonExpress II One Step Cloning Kit, Vazyme, China), resulting in the pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3 plasmid. The resulting colonies were screened for ampicillin resistance and confirmed by PCR and DNA sequencing of the amplified DNA fragment (Figure 5B, 5C).

Construction of Transgene K. xylinus
The plasmids pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3 and pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3 were each introduced into the sacB and gdh gene loci, respectively, via electroporation. However, we encountered a setback during the transformation process, as no colonies grew on the antibiotic selection plates, indicating a failure in electroporation.

Cycle 1 - Learn

Cycle 1 – Re-design

Cycle 1 – Re-build

Cycle 1 - Test

Cycle 1 - Learn

Shear force is a reason for the selection of spontaneous mutants, which can cause cellulose-producing (Cel+) cells to convert into non-cellulose-producing (Cel−) mutants [9]. To minimize the impact of shear force on BC production, we attempted to optimize the shaking speed and the viscosity of the fermentation broth by adding different concentrations of xanthan gum. However, reducing the shaking speed will inevitably affect bacterial growth rates, so we needed to determine an optimal speed.

Therefore, we were measuring the BC production of the pgi-OE gdh-KO strain at 50, 100, 150, and 200 r/min. Additionally, we tested the BC production of the pgi-OE gdh-KO strain in HS media with xanthan gum concentrations of 0%, 0.02%, 0.03%, and 0.05%.

No new components were constructed during this cycle.

All BC production measurements were taken on the seventh day after inoculation, with an initial inoculum size of 1%. We observed a significant impact of different shaking speeds on production yield. At a shaking speed of 150 r/min, the pgi-OE gdh-KO strain exhibited the highest yield, reaching a dry weight of 5.13 g/L (Figure 10A). At lower speeds, production decreased due to reduced growth rates; at higher speeds, the shear force in the fermentation broth caused a reduction in BC production.

Experiments increasing the viscosity of the fermentation broth showed that higher viscosity could improve cellulose production by approximately 10% (Figure 10B). However, once the xanthan gum concentration reached a certain level, it began to negatively affect BC yield. Previous studies have found that adding xanthan gum reduces the spaces between bacterial cellulose fibers, leading to a more compact structure [9].

We determined the optimal shaking speed and xanthan gum concentration for maximizing BC production. We added 0.03% xanthan gum to the HS medium to increase viscosity, resulting in a BC yield of 5.34 g/L. The optimal shaking speed was determined to be 150 r/min.

Selection of test trees

At the beginning, we were unfamiliar with the strategies for selecting tree species for afforestation in different regions. During our interviews with the Ant Forest public welfare organization, under the Alibaba Group, and desert botanists, they both recommended choosing local species that are drought-resistant and wind-resistant, preferably with some economic benefits. This led us to consider sea buckthorn.

Sea buckthorn (Hippophae rhamnoides) is highly drought-tolerant, with a deep and extensive root system that allows it to effectively absorb water and stabilize the soil, reducing water and soil erosion. Additionally, its strong adaptability to poor, sandy soils increases practical application potential.

BC (bacterial cellulose) possesses excellent physicochemical and mechanical properties, such as high purity, high crystallinity, strong water retention capacity, high degree of polymerization, large surface area, and good chemical stability. Additionally, compared to other water retention materials like hydrogels, polyacrylamide (PAM), and sodium carboxymethyl cellulose (CMC), BC offers biocompatibility, biodegradability, and renewability [10]. As a more cost-effective and sustainable solution, BC is particularly suitable for regions experiencing rapid desertification and severe soil moisture loss.

However, there is currently no data available on the water retention effects of bacterial cellulose (BC) in soil. Therefore, to mitigate potential environmental risks associated with our produced BC before it undergoes thorough scrutiny, we will use commercially available BC for the water retention tests.

To address this, we transplanted six sea-buckthorn trees, with three added to the soil with bacterial cellulose as the BC group and the other three without BC as the control group. Each had a similar initial growth state and a height of about 1 meter. The same weight of moist soil was placed in each pot. 200 g of wet bacterial cellulose was added to the soil of the BC group seedlings, while the control group soil did not contain BC. The soil distribution was as follows: the bottom layer consisted of soil with about 10% moisture, and the middle and upper layers were divided into those with and without BC, with soil moisture maintained at around 68%.

Starting with similar soil moisture levels, we withheld watering and continuously measured the soil moisture content, resulting in the following data:

At the start, the soil moisture content for all seedlings was around 68%. Over time, as water evaporated and was absorbed by the seedlings' roots, a clear difference in moisture retention between the groups became apparent. The BC group's water loss rate was noticeably slower than that of the control group. After 7 days, the BC group still maintained about 33% moisture content, while the control group's moisture content had dropped to 16.5%, which is below the minimum required for the seedlings to survive. Therefore, the application of BC during tree planting helps to slow soil moisture loss.

Impact of bacterial cellulose on seedling survival test

Due to limitations in venue and time, we were unable to conduct real-world tests of BC's impact on seedling survival rates in desertified areas. Consequently, we used mathematical modeling to predict the relationship between BC and seedling survival rates.

We employed a logistic regression model to predict the binary outcome of tree survival based on water retention rate and multiple other variables. The model's input variables included the main effects of each factor, as well as potential interaction and quadratic terms to capture nonlinear relationships. The estimated coefficients provided insight into the predicted probability of survival based on different conditions.

The logistic regression model can be expressed as follows:

where is the probability of tree survival, and is the linear combination of the input variables given by:

We demonstrated that BC helps slow the rate of moisture loss from the roots of seedlings, thereby improving the water retention capacity of the soil around the roots. In afforestation efforts aimed at combating desertification, enhanced water retention at the plant roots contributes to the survival rates of newly transplanted trees.

Moving forward, we plan to further optimize the genes in K. xylinus to achieve even higher BC yields. We also plan to apply BC to land with conditions more closely resembling deserts, to test its water retention capabilities and its effects on seedling survival rates. Moreover, we intend to investigate whether BC’s effectiveness varies across different tree species.

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