Simultaneous saccharification and fermentation (SSF) of pre-treated lignocellulosics to biofuels and other platform chemicals has long been a promising alternative to separate hydrolysis and fermentation processes. However, the disparity between the optimum conditions (temperature, pH) for fermentation and enzyme hydrolysis leads to execution of the SSF process at sub-optimal conditions, which can affect the rate of hydrolysis and cellulose conversion. The fermentation conditions could be synchronized with hydrolysis optima by carrying out the SSF at a higher temperature, but this would require a thermo-tolerant organism. Economically viable production of platform chemicals from lignocellulosic biomass (LCB) has long been stymied because of the significantly higher cost of hydrolytic enzymes. The major objective of this work is to develop an SSF strategy for D-lactic acid (D-LA) production by a thermo-tolerant organism, in which the enzyme loading could significantly be reduced without compromising on the overall conversion. A thermo-tolerant strain of Lactobacillus bulgaricus was developed by adaptive laboratory evolution (ALE) which enabled the SSF to be performed at 45 °C with reduced enzyme usage. Despite the reduction of enzyme loading from 15 Filter Paper Unit/gLCB (FPU/gLCB) to 5 FPU/gLCB, we could still achieve ~ 8% higher cellulose to D-LA conversion in batch SSF, in comparison to the conversion by separate enzymatic hydrolysis and fermentation processes at 45 °C and pH 5.5. Extending the batch SSF to SSF with pulse-feeding of 5% pre-treated biomass and 5 FPU/gLCB, at 12-h intervals (36th–96th h), resulted in a titer of 108 g/L D-LA and 60% conversion of cellulose to D-LA. This is one among the highest reported D-LA titers achieved from LCB. We have demonstrated that the SSF strategy, in conjunction with evolutionary engineering, could drastically reduce enzyme requirement and be the way forward for economical production of platform chemicals from lignocellulosics. We have shown that fed-batch SSF processes, designed with multiple pulse-feedings of the pre-treated biomass and enzyme, can be an effective way of enhancing the product concentrations.

中文翻译：

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保加利亚乳杆菌的进化工程通过同时糖化和发酵减少酶的使用并增强木质纤维素向 D-乳酸的转化

将预处理的木质纤维素同时糖化和发酵 (SSF) 制成生物燃料和其他平台化学品长期以来一直是分离水解和发酵过程的有希望的替代方案。然而，发酵和酶水解的最佳条件（温度、pH）之间的差异导致 SSF 工艺在次优条件下执行，这会影响水解和纤维素转化的速率。通过在较高温度下进行 SSF，发酵条件可以与最适水解同步，但这需要耐热的生物体。长期以来，由于水解酶成本显着升高，从木质纤维素生物质 (LCB) 生产平台化学品的经济可行性一直受到阻碍。这项工作的主要目的是开发一种由耐热生物生产 D-乳酸 (D-LA) 的 SSF 策略，其中酶负荷可以显着降低而不影响整体转化。通过适应性实验室进化 (ALE) 开发了一种耐热的保加利亚乳杆菌菌株，该菌株使 SSF 能够在 45°C 下进行，同时减少酶的使用。尽管酶载量从 15 个滤纸单位/gLCB (FPU/gLCB) 减少到 5 FPU/gLCB，与单独的酶促转化相比，我们仍然可以在批量 SSF 中实现约 8% 的纤维素转化为 D-LA在 45 °C 和 pH 5.5 条件下进行水解和发酵过程。将批次 SSF 扩展到 SSF，以 12 小时间隔（第 36-96 小时）脉冲进料 5% 预处理生物质和 5 FPU/gLCB，导致 108 g/L D-LA 的滴度和 60% 的纤维素转化为 D-LA。这是从 LCB 获得的最高报告的 D-LA 滴度之一。我们已经证明，SSF 策略与进化工程相结合，可以大大降低酶的需求，并成为从木质纤维素中经济地生产平台化学品的前进方向。我们已经表明，采用预处理生物质和酶的多次脉冲进料设计的分批补料 SSF 工艺可以成为提高产品浓度的有效方法。可以大大降低酶的需求，并成为从木质纤维素经济地生产平台化学品的前进方向。我们已经表明，采用预处理生物质和酶的多次脉冲进料设计的分批补料 SSF 工艺可以成为提高产品浓度的有效方法。可以大大降低酶的需求，并成为从木质纤维素经济地生产平台化学品的前进方向。我们已经表明，采用预处理生物质和酶的多次脉冲进料设计的分批补料 SSF 工艺可以成为提高产品浓度的有效方法。