Production of Propionate by a Sequential Fermentation-Biotransformation Process via l-Threonine.

Bio-based propionate is widely welcome in the food additive industry. The current anaerobic process by Propionibacteria endures low titers and a long fermentation time. In this study, a new route for propionate production from l-threonine was designed. 2-Ketobutyrate, deaminated from l-threonine, is cleaved into propionaldehyde and CO2 and then be oxidized into propionic acid, which is neutralized by ammonia released from the first deamination step. This CoA-independent pathway with only CO2 as a byproduct boosts propionate production from l-threonine with high productivity and purity. The key enzyme for 2-ketobutyrate decarboxylation was selected, and its expression was optimized. The engineered Pseudomonas putida strain, harboring 2-ketoisovalerate decarboxylase from Lactococcus lactis could produce 580 mM (43 g/L) pure propionic acid from 600 mM l-threonine in 24 h in the batch biotransformation process. Furthermore, a high titer of 62 g/L propionic acid with a productivity of 1.07 g/L/h and a molar yield of >0.98 was achieved in the fed-batch pattern. Finally, an efficient sequential fermentation-biotransformation process was demonstrated to produce propionate directly from the fermentation broth containing l-threonine, which further reduces the costs since no l-threonine purification step is required.

Highly efficient production of L-homoserine in Escherichia coli by engineering a redox balance route.

L-Homoserine is a nonessential chiral amino acid and the precursor of L-threonine and L-methionine. It has great potential to be used in the pharmaceutical, agricultural, cosmetic, and fragrance industries. However, the current low efficiency in the fermentation process of L-homoserine drives up the cost and therefore limits applications. Here, we systematically analyzed the L-homoserine production network in Escherichia coli to design a redox balance route for L-homoserine fermentation from glucose. Production of L-homoserine from L-aspartate via reduction of the tricarboxylic acid cycle intermediate oxaloacetate lacks reducing power. This deficiency could be corrected by activating the glyoxylate shunt and driving the flux from fumarate to L-aspartate with excess reducing power. This redox balance route decreases cell growth pressure and the theoretical yield of L-homoserine is 1.5 mol/mol of glucose without carbon loss. We fine-tuned the flux from fumarate to L-aspartate, deleted competitive and degradative pathways, enhanced L-homoserine efflux, and generated 84.1 g/L L-homoserine with 1.96 g/L/h productivity and 0.50 g/g glucose yield in a fed-batch fermentation. This study proposes a novel balanced redox metabolic network strategy for highly efficient production of L-homoserine and its derivative amino acids.

[Metabolic regulation in constructing microbial cell factories].

The regulation of the expression of genes involved in metabolic pathways, termed as metabolic regulation, is vital to construct efficient microbial cell factories. With the continuous breakthroughs in synthetic biology, the mining and artificial design of high-quality regulatory elements have substantially improved our ability to modify and regulate cellular metabolic networks and its activities. The research on metabolic regulation has also evolved from the static regulation of single genes to the intelligent and precise dynamic regulation at the systems level. This review briefly summarizes the advances of metabolic regulation technologies in the past 30 years.

Unravelling the thioesterases responsible for propionate formation in engineered Pseudomonas putida KT2440.

Pseudomonas putida KT2440 is becoming a new robust metabolic chassis for biotechnological applications, due to its metabolic versatility, low nutritional requirements and biosafety status. We have previously engineered P. putida KT2440 to be an efficient propionate producer from L-threonine, although the internal enzymes converting propionyl-CoA to propionate are not clear. In this study, we thoroughly investigated 13 genes annotated as potential thioesterases in the KT2440 mutant. One thioesterase encoded by locus tag PP_4975 was verified to be the major contributor to propionate production in vivo. Deletion of PP_4975 significantly decreased propionate production, whereas the performance was fully restored by gene complement. Compared with thioesterase HiYciA from Haemophilus influenza, thioesterase PP_4975 showed a faster substrate conversion rate in vitro. Thus, this study expands our knowledge on acyl-CoA thioesterases in P. putida KT2440 and may also reveal a new target for further engineering the strain to improve propionate production performance.

One major facilitator superfamily transporter is responsible for propionic acid tolerance in Pseudomonas putida KT2440.

Propionic acid (PA) has been widely used as a food preservative and chemical intermediate in the agricultural and pharmaceutical industries. Environmental and friendly biotechnological production of PA from biomass has been considered as an alternative to the traditional petrochemical route. However, because PA is a strong inhibitor of cell growth, the biotechnological host should be not only able to produce the compound but the host should be robust. In this study, we identified key PA tolerance factors in Pseudomonas putida KT2440 strain by comparative transcriptional analysis in the presence or absence of PA stress. The identified major facilitator superfamily (MFS) transporter gene cluster of PP_1271, PP_1272 and PP_1273 was experimentally verified to be involved in PA tolerance in P. putida strains. Overexpression of this cluster improved tolerance to PA in a PA producing strain, what is useful to further engineer this robust platform not only for PA synthesis but for the production of other weak acids.

Bio-production of high-purity propionate by engineering L-threonine degradation pathway in Pseudomonas putida.

Propionic acid (PA) is widely used in the food, agricultural, and pharmaceutical industries. Since the petrochemical PA is unsustainable, biological production of PA from renewable substrates is gaining attention. In this study, we engineered the strain Pseudomonas putida KT2440 to transform L-threonine to PA with only CO2 released as by-product. The cell factory was created by chromosomal incorporation of heterologous L-threonine deaminase, permease, and acyl-CoA thioesterase, deletion of branch pathways as well as overproduction of the endogenous branched-chain alpha-keto acid dehydrogenase complex. The final engineered strain could produce 399 mM PA from 400 mM L-threonine in a batch biotransformation process, with a molar yield of 99.8% under the optimized conditions in 48 h. The PA titer further reached to 50.3 g/L (679 mM) with a productivity of 0.6 g/L/h in a fed-batch conversion process. No obvious by-products, such as acetate and succinate, were detected in the broth, which would significantly facilitate downstream purification steps. Thus, this study offers an alternative route for biological production of PA.

Thermostability improvement of the glucose oxidase from Aspergillus niger for efficient gluconic acid production via computational design.

Gluconic acid (GA) and its alkali salts are extensively used in the food, feed, beverage, textile, pharmaceutical and construction industries. However, the cost-effective and eco-friendly production of GA remains a challenge. The biocatalytic process involving the conversion of glucose to GA is catalysed by glucose oxidase (GOD), in which the catalytic efficiency is highly dependent on the GOD stability. In this study, we used in silico design to enhance the stability of glucose oxidase from Aspergillus niger. A combination of the best mutations increased the apparent melting temperature by 8.5 °C and significantly enhanced thermostability and thermoactivation. The variant also showed an increased optimal temperature without compromising the catalytic activity at lower temperatures. Moreover, the combined variant showed higher tolerance at pH 6.0 and 7.0, at which the wild-type enzyme rapidly deactivated. For GA production, an approximate 2-fold higher GA production yield was obtained, in which an almost complete conversion of 324 g/L d-glucose to GA was achieved within 18 h. Collectively, this work provides novel and efficient approaches for improving GOD thermostability, and the obtained variant constructed by the computational strategy can be used as an efficient biocatalyst for GA production at industrially viable conditions.

[High-level production of glucose oxidase by recombinant Pichia pastoris using a combined strategy].

To enhance the production of glucose oxidase by recombinant Pichia pastoris, two strategies were developed, which were namely co-feeding of methanol and sorbitol and co-expressing of the protein disulfide isomerase (PDI) and Vitreoscialla hemoglobin (VHb). The volumetric activity reached 456 U/mL by using the strain X33/pPIC9k-GOD, in 5 liter fermentator, with the co-feeding of methanol and sorbitol, it was 0.2 fold higher than that only feeding by methanol. The improved strain was obtained by co-expressing PDI-VHb with GOD. While fermented in a 5 liter fermentator by feeding methanol and sorbitol, the activity of the improved strain reached 716 U/mL with a yield of 7 400 mg/L total soluble protein concentration. These results indicated that heterologous protein expression level can be enhanced by optimizing fermentation condition and co-expression molecular chaperon in Pichia pastoris.